This invention relates to a purified nucleotide sequence coding for a repressor protein for regulating gene expression. In addition, this invention relates to a recombinant expression vector containing the nucleotide sequence, an E. coli cell transformed or transfected with the recombinant expression vector, and the use of the E. coli cells for preparing Pseudomonas cepacia 2,2-dialkylglycine decarboxylase. Further, this invention provides a repressor protein encoded by the nucleotide sequence, wherein the repressor protein is in a purified form.
The 2,2-dialkylglycine decarboxylase of the soil bacterium Pseudomonas cepacia was first reported by Aaslestad and Larson (1964) and was later investigated in several laboratories (Bailey and Dempsey, 1967; Bailey et al., 1970; Lamartiniere et al., 1971; Honma et al., 1972; Sato et al., 1978; and Keller and O'Leary, 1979). This pyridoxal 5'-phosphate-dependent enzyme catalyzes decomposition of substrate amino acids such as 2-methylalanine and isovaline in two steps: (i) release of carbon dioxide and ketone with transfer of the amino group to the cofactor to give enzyme-bound pyridoxamine 5'-phosphate and (ii) amino transfer from cofactor to pyruvate forming L-alanine and regenerating the cofactor in the aldehyde oxidation state. The decarboxylation step is analogous to the so-called abortive decarboxylation catalyzed by several pyridoxal 5'-phosphate-dependent amino acid decarboxylases, which competes with the normal hydrogen for carboxylate replacement reaction (Sukhareva, 1986). The dialkylglycine decarboxylase is of interest because it normally catalyzes both decarboxylation and amino transfer. Therefore, the question arises whether this enzyme is an aminotransferase that through evolution has added a decarboxylase capability or is a decarboxylase that has evolved an amino transfer capability. A preliminary answer as provided to this question by showing that the dialkylglycine decarboxylase primary structure is homologous to several aminotransferases but not to decarboxylases.
The biological role of the dialkylglycine decarboxylase remains unclear. The substrates 2-methylalanine and isovaline occur naturally as major constituents of cytotoxic peptides produced by soil fungi such as Trichoderma viride (Bruckner et al., 1980; Bruckner and Pryzbylaki, 1984; Schmitt and Jung, 1985) and as organic components of carbonaceous meteorites (Kvenvolden et al., 1971). Racemic isovaline and 2-methylalanine have been found recently in an iridium-rich Cretaceous-Tertiary boundary layer, further supporting an extraterrestrial source for this material (Zhao and Bada, 1989). Thus, the enzyme may have evolved to use the rare dialkylglycines of cosmic origin, or it may be a part of a metabolic pathway for breaking down cytotoxic peptides and the constituent amino acids.
The available structural information about the 2,2-dialkylglycine decarboxylase is sparse. Lamartiniere et al. (1971) showed by equilibrium sedimentation that a dialkylglycine decarboxylase isolated from P. cepacia has a molecular mass of 188 kDa with four identical 47-kDa subunits. They also reported a peptide map and amino acid composition data consistent with a 47-kDa subunit. Sato et al, (1978) also studied the P. cepacia dialkylglycine decarboxylase, showing by gel electrophoresis that the 180-kDa holoenzyme contained four identical subunits of approximately 45 kDa and presenting chemical labeling evidence for a catalytically important histidine residue.